Disturbances in circadian rhythms are known to contribute to a variety of diseases and to impair mental and physical performance. Within individual suprachiasmatic nucleus (SCN) neurons, circadian rhythms are generated by molecular clocks consisting of gene transcription feedback loops. Yet not all SCN neurons have rhythmic clock gene expression, a finding that raises the question of how neurons with clocks communicate with each other and with those neurons lacking clocks to generate circadian output. This lack of knowledge is a major impediment to understanding how cellular clocks govern circadian behavior. In mammals, these rhythmic and non-rhythmic neurons are organized into anatomical compartments in the SCN, which is the master circadian oscillator. These compartments differ in clock gene expression patterns, as well as in efferent and afferent neuronal connections. The long-term goal of our research is to characterize SCN compartments and their functional significance. Our current goal is to determine the synaptic signaling mechanisms of phenotypically identified neurons in a defined SCN compartment, the calbindin sub-nucleus (CBsn), which is known to be important for generating circadian locomotor behavior. Our hypothesis is that the generation of circadian outputs depends on the micro-circuitry within and between SCN compartments. To test this, we will use a unique combination of electrophysiological recording and immunohistochemical staining techniques to characterize synaptic transmission in the CBsn. Because we are using a defined cell population and a novel combination of techniques, we expect to be able to draw strong conclusions about the micro-circuitry of the CBsn. The specific aims of the proposal are: 1) Determine the circadian phase dependence of responses of CBsn neurons to optic nerve stimulation. 2) Characterize the regulation of action potential firing and synaptic transmission by vasoactive intestinal peptide (VIP) and pituitary adenyl cyclase activating polypeptide (PACAP). 3) Examine the responses of CBsn neurons to Transforming Growth Factor alpha (TGFalpha). 4) Determine whether gastrin releasing peptide (GRP) or VIP immunoreactive neurons fire action potentials in a circadian manner. Completion of these studies will lead to a better understanding of the cellular basis of circadian rhythms as well as the potential to better treat sleep disorders and other disturbances in the circadian clock.